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Proton trapping in the cellular acidic vacuolar compartment: lysosomal mechanisms in apoptosis/necrosis and iron chelation
Linköping University, Department of Neuroscience and Locomotion, Neurosurgery. Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Under ischemic conditions, a number of cytotoxic metabolic products are formed. Reactive oxygen species are known to be important mediators of progressive ischemic cell injury, and the synergistic damage to cells caused by the combination of such oxygen species and redox-active iron is well appreciated. The acidic interior of lysosome leads to the trapping of substances with high pK4 values. A large variety of molecules, being weak bases, may thus concentrate within this acidic vacuolar compartment, potentially leading to both beneficial and detrimental effects. A major part of the intracellular pool of redoxactive iron is likely to be located in the lysosomal compartment, and iron chelators that are lysosomotropic due to high pK4 values may prove to be important pharmacological tools to protect the brain from oxidative stress. Among a variety of substances formed in the ischemic penumbra zone is the polyamine metabolite, 3-aminopropanal (3-AP), a substance of extreme neurotoxicity. 3-AP is a weak base and may theoretically exert its toxic action through induction of cell death after intralysosomal accumulation.

On the 1774 mouse histiocytic lymphoma cell line, we used the common lysosomotropic agent NH3 to increase lysosomal pH, the lysosomotropic iron chelator, 5-[1,2] dithiolan-3-yl-pentanoic acid (2-dimethylamino-ethyl)-amide (LAP) and the lysosomotropic iron binder, WR-1065, a metabolite of amifostine, as tools to determine that proton trapping within the lysosomal acidic vacuolar compartment plays an important role in oxidative stress-induced apoptosis. We also used another lysosomotropic agent, 3-AP, on the J774 cell line and on the SH-SY5Y human neuroblastoma cell line. The results indicate that proton trapping of this toxin within the lysosome might explain its toxicity to cells.

Sulfide-silver cytochemical detection of iron revealed a pronounced decrease in the lysosomal content of redox-active iron following reduced acidity of the lysosome, and electron spin-resonance studies showed that no hydroxyl radicals [OH] were formed from hydrogen peroxide under these conditions. This suggests that lysosomes contain most of the free, redox-active iron. In further support of this idea, the lysosomotropic agents LAP and WR-1065 were found to be 5000 and 2500 times more effective, respectively, in protecting cells from oxidative stress, compared with the well-known iron chelator desferrioxamine [DFO]. Evidence was obtained that LAP and WR-1065 exerted their effect on intralysosomal redox-active iron, and that the effect was linked to the acidity of the lysosome. Being weak bases (LAP, pKa = 8.0; WR-1065, pKa = 9.2), these compounds accumulate intralysosomally by proton trapping. The neurotoxic effect of 3-AP (pKa = 9.3) could be linked to a dose-dependent induction of cell death, most likely based on intralysosomal proton trapping of this molecule followed by lysosomal rupture. The lysosomal rupture seems to induce a chain of intracellular events (including generation of oxidative stress), leading to mitochondrial damage directly or indirectly caused by the release of lysosomal proteases.

We conclude that the low pH of the lysosome may both serve to attract basic toxins, such as 3-AP, and promote the accumulation of protective agents, such as LAP and WR-1065. Prevention of lysosomal damage from both oxidants and neurotoxins by lysosomotropic agents has great potential therapeutic utility.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2003. , 58 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 808
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-28088Local ID: 12854ISBN: 91-7373-501-9 (print)OAI: oai:DiVA.org:liu-28088DiVA: diva2:248639
Public defence
2003-10-09, Patologens föreläsningssal, Universitetssjukhuset, Linköping, 13:15 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-16Bibliographically approved
List of papers
1. Intralysosomal iron: a major determinant of oxidant-induced cell death
Open this publication in new window or tab >>Intralysosomal iron: a major determinant of oxidant-induced cell death
2003 (English)In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 34, no 10, 1243-1252 p.Article in journal (Refereed) Published
Abstract [en]

As a result of continuous digestion of iron-containing metalloproteins, the lysosomes within normal cells contain a pool of labile, redox-active, low-molecular-weight iron, which may make these organelles particularly susceptible to oxidative damage. Oxidant-mediated destabilization of lysosomal membranes with release of hydrolytic enzymes into the cell cytoplasm can lead to a cascade of events eventuating in cell death (either apoptotic or necrotic depending on the magnitude of the insult). To assess the importance of the intralysosomal pool of redox-active iron, we have temporarily blocked lysosomal digestion by exposing cells to the lysosomotropic alkalinizing agent, ammonium chloride (NH4Cl). The consequent increase in lysosomal pH (from ca. 4.5 to > 6) inhibits intralysosomal proteolysis and, hence, the continuous flow of reactive iron into this pool. Preincubation of J774 cells with 10 mM NH4Cl for 4 h dramatically decreased apoptotic death caused by subsequent exposure to H2O2, and the protection was as great as that afforded by the powerful iron chelator, desferrioxamine (which probably localizes predominantly in the lysosomal compartment). Sulfide-silver cytochemical detection of iron revealed a pronounced decrease in lysosomal content of redox-active iron after NH4Cl exposure, probably due to diminished intralysosomal digestion of iron-containing material coupled with continuing iron export from this organelle. Electron paramagnetic resonance experiments revealed that hydroxyl radical formation, readily detectable in control cells following H2O2 addition, was absent in cells preexposed to 10 mM NH4Cl. Thus, the major pool of redox-active, low-molecular-weight iron may be located within the lysosomes. In a number of clinical situations, pharmacologic strategies that minimize the amount or reactivity of intralysosomal iron should be effective in preventing oxidant-induced cell death.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27102 (URN)10.1016/S0891-5849(03)00109-6 (DOI)11749 (Local ID)11749 (Archive number)11749 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
2. Prevention of oxidant-induced cell death by lysosomotropic iron chelators
Open this publication in new window or tab >>Prevention of oxidant-induced cell death by lysosomotropic iron chelators
Show others...
2003 (English)In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 34, no 10, 1295-1305 p.Article in journal (Refereed) Published
Abstract [en]

Intralysosomal iron powerfully synergizes oxidant-induced cellular damage. The iron chelator, desferrioxamine (DFO), protects cultured cells against oxidant challenge but pharmacologically effective concentrations of this drug cannot readily be achieved in vivo. DFO localizes almost exclusively within the lysosomes following endocytic uptake, suggesting that truly lysosomotropic chelators might be even more effective. We hypothesized that an amine derivative of α-lipoamide (LM), 5-[1,2] dithiolan-3-yl-pentanoic acid (2-dimethylamino-ethyl)-amide (α-lipoic acid-plus [LAP]; pKa = 8.0), would concentrate via proton trapping within lysosomes, and that the vicinal thiols of the reduced form of this agent would interact with intralysosomal iron, preventing oxidant-mediated cell damage. Using a thiol-reactive fluorochrome, we find that reduced LAP does accumulate within the lysosomes of cultured J774 cells. Furthermore, LAP is approximately 1,000 and 5,000 times more effective than LM and DFO, respectively, in protecting lysosomes against oxidant-induced rupture and in preventing ensuing apoptotic cell death. Suppression of lysosomal accumulation of LAP (by ammonium-mediated lysosomal alkalinization) blocks these protective effects. Electron paramagnetic resonance reveals that the intracellular generation of hydroxyl radical following addition of hydrogen peroxide to J774 cells is totally eliminated by pretreatment with either DFO (1 mM) or LAP (0.2 μM) whereas LM (200 μM) is much less effective.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27105 (URN)10.1016/S0891-5849(03)00106-0 (DOI)11752 (Local ID)11752 (Archive number)11752 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
3. The radioprotective agent, amifostine, suppresses the reactivity of intralysosomal iron
Open this publication in new window or tab >>The radioprotective agent, amifostine, suppresses the reactivity of intralysosomal iron
2003 (English)In: Redox report, ISSN 1351-0002, E-ISSN 1743-2928, Vol. 8, no 6, 347-355 p.Article in journal (Refereed) Published
Abstract [en]

Amifostine (2-[(3-aminopropyl)amino]ethane-thiol dihydrogen phosphate ester; WR-2721) is a radioprotective agent used clinically to minimize damage from radiation therapy to adjacent normal tissues. This inorganic thiophosphate requires dephosphorylation to produce the active, cell-permeant thiol metabolite, WR-1065. The activation step is presumably catalyzed by membrane-bound alkaline phosphatase, activity of which is substantially higher in the endothelium of normal tissues. This site-specific delivery may explain the preferential protection of normal versus neoplastic tissues. Although it was developed several decades ago, the mechanisms through which this agent exerts its protective effects remain unknown. Because WR-1065 is a weak base (pKa = 9.2), we hypothesized that the drug should preferentially accumulate (via proton trapping) within the acidic environment of intracellular lysosomes. These organelles contain abundant 'loose' iron and represent a likely initial target for oxidant- and radiation-mediated damage. We further hypothesized that, within the lysosomal compartment, the thiol groups of WR-1065 would interact with this iron, thereby minimizing iron-catalyzed lysosomal damage and ensuing cell death. A similar mechanism of protection via intralysosomal iron chelation has been invoked for the hexadentate iron chelator, desferrioxamine (DFO; although DFO enters the lysosomal compartment by endocytosis, not proton trapping). Using cultured J774 cells as a model system, we found substantial accumulation of WR-1065 within intracellular granules as revealed by reaction with the thiol-binding fluorochrome, BODIPY FL L-cystine. These granules are lysosomes as indicated by co-localization of BODIPY staining with LysoTracker Red. Compared to 1 mM DFO, cells pre-treated with 0.4 ?M WR-1065 are protected from hydrogen peroxide-mediated lysosomal rupture and ensuing cell death. On a molar basis in this experimental system, WR-1065 is approximately 2500 times more effective than DFO in preventing oxidant-induced lysosomal rupture and cell death. This increased effectiveness is most likely due to the preferential concentration of this weak base within the acidic lysosomal apparatus. By electron spin resonance, we found that the generation of hydroxyl radical, which normally occurs following addition of hydrogen peroxide to J774 cells, is totally blocked by pretreatment with either WR-1065 or DFO. These findings suggest a single and plausible explanation for the radioprotective effects of amifostine and may provide a basis for the design of even more effective radio- and chemoprotective drugs.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27104 (URN)10.1179/135100003225003384 (DOI)11751 (Local ID)11751 (Archive number)11751 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
4. 3-Aminopropanal is a lysosomotropic aldehyde that causes oxidative stress and apoptosis by rupturing lysosomes
Open this publication in new window or tab >>3-Aminopropanal is a lysosomotropic aldehyde that causes oxidative stress and apoptosis by rupturing lysosomes
2003 (English)In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 111, no 6, 643-652 p.Article in journal (Refereed) Published
Abstract [en]

During cerebral ischemia and following trauma, potent cytotoxic polyamine-derived aminoaldehydes form, diffuse, and damage adjacent tissues not directly subjected to the initial insult. One such aldehyde is 3-aminopropanal (3-AP). The mechanisms by which such a small aldehydic compound is excessively cytotoxic have been unclear until recently when we showed that 3-AP, having the structure of a weak lysosomotropic base, concentrates within the acidic vacuolar compartment and causes lysosomal rupture that, in turn, induces caspase activation and apoptotic cell death. Here, using cultured J774 cells and 3-AP as a way to selectively burst lysosomes, we show that moderate lysosomal rupture induces a transient wave of oxidative stress. The start of this oxidative stress period is concomitant with a short period of enhanced mitochondrial membrane potential that later fades and is replaced by a decreased potential before the oxidative stress diminishes. The result of the study suggests that oxidative stress, which has often been described during apoptosis induced by agonists other than oxidative stress per se, may be a consequence of lysosomal rupture with direct and/or indirect effects on mitochondrial respiration and electron transport causing a period of passing enhanced formation of reactive oxygen species.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27708 (URN)10.1034/j.1600-0463.2003.1110607.x (DOI)12446 (Local ID)12446 (Archive number)12446 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
5. Human neuroblastoma (SH-SY5Y) cells are highly sensitive to the lysosomotropic aldehyde 3-aminopropanal
Open this publication in new window or tab >>Human neuroblastoma (SH-SY5Y) cells are highly sensitive to the lysosomotropic aldehyde 3-aminopropanal
2004 (English)In: Brain Research, ISSN 0006-8993, E-ISSN 1872-6240, Vol. 1016, no 2, 163-169 p.Article in journal (Refereed) Published
Abstract [en]

3-Aminopropanal (3-AP), a degradation product of polyamines such as spermine, spermidine and putrescine, is a lysosomotropic small aldehyde that causes apoptosis or necrosis of most cells in culture, apparently by inducing moderate or extensive lysosomal rupture, respectively, and secondary mitochondrial changes. Here, using the human neuroblastoma SH-SY5Y cell line, we found simultaneous occurrence of apoptotic and necrotic cell death when cultures were exposed to 3-AP in concentrations that usually are either nontoxic, or only cause apoptosis. At 30 mM, but not at 10 mM, the lysosomotropic base and proton acceptor NH3 completely blocked the toxic effect of 3-AP, proving that 3-AP is lysosomotropic and suggesting that the lysosomal membrane proton pump of neuroblastoma cells is highly effective, creating a lower than normal lysosomal pH and, thus, extensive intralysosomal accumulation of lysosomotropic drugs. A wave of internal oxidative stress, secondary to changes in mitochondrial membrane potential, followed and gave rise to further lysosomal rupture. The preincubation of cells for 24 h with a chain-breaking free radical-scavenger, α-tocopherol, before exposure to 3-AP, significantly delayed both the wave of oxidative stress and the secondary lysosomal rupture, while it did not interfere with the early 3-AP-mediated phase of lysosomal break. Obviously, the reported oxidative stress and apoptosis/necrosis are consequences of lysosomal rupture with ensuing release of lysosomal enzymes resulting in direct/indirect effects on mitochondrial permeability, membrane potential, and electron transport. The induced oxidative stress seems to act as an amplifying loop causing further lysosomal break that can be partially prevented by α-tocopherol. Perhaps secondary brain damage during a critical post injury period can be prevented by the use of drugs that temporarily raise lysosomal pH, inactivate intralysosomal 3-AP, or stabilize lysosomal membranes against oxidative stress.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-23792 (URN)10.1016/j.brainres.2004.04.075 (DOI)3310 (Local ID)3310 (Archive number)3310 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved

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